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ELIZABETH NOLAN:
Welcome to the class.

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We're going to discuss
the themes that

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are going to basically
permeate every topic

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and module we'll
talk about here.

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And one of the central
themes of this class

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is that we're
interested in studying

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the cellular processes of life
at a molecular level, right?

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So as biochemists
and chemists, we're

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interested in this
level of understanding.

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And what we see here is a
cartoon depiction of the cell.

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And we see that there's
many types of biomolecules

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in this environment.

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So what are our core
themes for this year?

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First, we believe that life must
be studied on a molecular level

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to truly understand it.

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And so we need to think about
the cellular environment,

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both on a macroscopic scale,
and on the molecular level.

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And this environment is
complex, and it always

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needs to be considered, right?

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So as experimentalists
in biochemistry, often

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we're doing experiments
in aqueous buffer

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with proteins or some
other biomolecule.

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How does that relate to
a context like this one

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here where the environment is
very different and much more

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complex?

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Something we'll see,
especially in the first half,

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the first four modules
of this course,

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is that in cells,
complex processes

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are carried out
by macromolecular

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machines and elaborate systems.

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And these systems
are fascinating.

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You'll see that we know a
lot, but as we learn more,

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there's more and more
questions that come up,

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and more questions we
need to address with that.

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In addition to these
macromolecular machines,

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some additional
themes for this course

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involve homeostasis
and signaling.

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And these will be
especially emphasized

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in the second half of the course
when Professor Stubbe takes

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over there.

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So how do we think about
homeostasis and signaling

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in these contexts?

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Something that will
come up again and again

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is how, basically,
understanding cellular processes

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at a molecular level, or
the molecular features,

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can help explain mechanisms
of human disease,

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as well as therapeutics.

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So an example we'll see in
the early part of this lecture

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involves the ribosome.

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So many antibiotics
target the ribosome.

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And by understanding ribosome
structure and function,

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we can understand how these
small molecule therapeutics

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work.

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Another example
involves the proteasome

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which we'll hear about in the
second half of the course.

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So there's therapeutics
that target the proteasome,

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for instance, for cancer.

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And cholesterol
biosynthesis will come up,

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and how does our understanding
of cholesterol biosynthesis

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lead to ways to treat
coronary disease?

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Something that
JoAnne and I really

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like to think about
day-to-day and convey

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to you in this course
is the importance

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of experimental design,
and choice of methods.

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So as scientists and
experimentalists,

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how do we think about
designing an experiment,

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because that design is really
critical to the outcome,

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and what we can
make of the data?

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And so throughout
lectures and recitations,

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things to keep in mind,
and that we'll reiterate,

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are that all techniques
have inherent strengths

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and limitations.

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And so it's
something we all need

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to keep in mind
when we analyze data

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and think about how an
experiment was done.

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And these systems we're
going to look at in 5.08

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are very complex.

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And what that means is
that many different types

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of experimental
method are needed

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in order to answer complex--

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and sometimes not so complex--

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questions.

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So one method alone
just often isn't enough.

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We need insights from many
different techniques and types

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of expertise.

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And so we look forward to
informing you about different

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types of methods-- whether they
be established and quite old

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or new--

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that are important today.

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And as I alluded to
before, something

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we have to keep in mind when
doing biochemistry in the lab

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is that the test tube is
very different from the cell.

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These environments
are vastly different,

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and so we always need to think
about how to relate data back

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to a cellular or
physiological context.

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If you measure a dissociation
constant of one micromolar,

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what does that mean in a
cell versus one picomolar,

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for instance.

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Another point to make
is that the hypothesis

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is a moving target.

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So we have the
hypothesis, experiments

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are designed to test
this hypothesis,

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and there's some outcome.

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Maybe that supports the
hypothesis, maybe not.

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Or maybe there's
some new insight

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from a related field
that really changes

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how we think about something.

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So in many cases
we're integrating

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data and insights that are
quite new, and Professor Stubbe

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and I won't have
all of the answers.

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And so that type of
uncertainty is something

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that we aim for you all to gain
some level of comfort with.

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So there's many complexities
in primary data, often

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uncertainties.

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And that's just an
aspect of this course.

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And scientists, it's
something we grapple

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with every day in our own work.

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So we're introducing
that to you here.

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And along those lines, just
keep in mind, we know so much.

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And I think it's amazing,
and-- if I step back and think

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about this for
some of the systems

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we'll see-- actually
overwhelming.

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And it's really due to dedicated
efforts of many, many people

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over many, many years.

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But with that said, there are
so many remaining unanswered

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questions, and we hope that
you'll find inspiration

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in some of these questions
as looking forward

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within this field.

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There.

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OK, so what about the cell
and macromolecular crowding?

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Just to emphasize
this point a bit more,

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here we have an E. coli.

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OK, so E. coli are laboratory
workhorses for biochemists.

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They're fascinating,
I love E. coli.

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But I just show you this
simple E. coli cartoon

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and this depiction here
to emphasize how crowded

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the cellular environment is.

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So we have an equal E. coli
of about two microns long,

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and maybe half a micron wide,
a volume of about a femtolitre.

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And if we think about the
E. coli genome for a minute,

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it encodes about 4,000 proteins.

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That's a lot of proteins.

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And if we think about one E.
coli cell of this small size,

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can just ask a simple question,
how many ribosomes are there?

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So we all know the
ribosomes are needed

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for polypeptide biosynthesis.

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How many ribosomes are
packaged in one E. coli?

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Any guess?

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So, 10, 100, a million.

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AUDIENCE: Order of 1,000?

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ELIZABETH NOLAN: Pardon?

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AUDIENCE: Order of like, 1,000?

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ELIZABETH NOLAN: Yeah, let's
say 1,000 times 15 or 20.

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So there's about 15,000
to 20,000 ribosomes

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in one E. coli cell.

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And as we'll see in
Friday's lecture,

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the ribosome is very large.

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How did they all fit?

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And there's not
only the ribosomes,

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but there's many,
many other players,

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just as noted here
in this cartoon.

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So you can think about
what does that mean

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in terms of concentrations.

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We'll bring up concentrations
of biomolecules in the cell

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throughout this course,
and what does it

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mean having them packaged
together so much here?

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So, very different
than the test tube.

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Our goals, some of
which I think have

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been communicated by me so far.

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But just to emphasize,
we're interested

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in these macromolecular
machines and chemical

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processes responsible for life.

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We hope by the end
of this course,

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everyone gains an appreciation
for the complexity of life,

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and our current
understanding of the topics

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we present to you this spring.

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There's close links between
basic fundamental research

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and medicine, and technology
development as well.

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Understanding the
experimental basis

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for understanding,
methods and hypotheses.

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And what we think is something
that we hope to achieve,

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and that you can
bring to other places

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after this course is really
to be able to knowledgeably

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and critically evaluate
methods and results, especially

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primary data.

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And we also hope
that we convince you

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that biological
chemistry is really

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thought provoking
and fun, and hope

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you all think that
right now as well.

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So what are the actual
topics we're going to cover?

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We organized this
course into modules,

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and these modules
are listed here.

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And different modules will
have different numbers

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00:10:00,170 --> 00:10:02,360
of lectures dedicated to them.

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00:10:02,360 --> 00:10:06,320
But where we'll go between
now and spring break--

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I'll present to you
during these weeks--

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is that we're going to focus
on the lifecycle of a protein

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for the first three modules.

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And many of you are familiar
with aspects of this.

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We're going to
present these topics,

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I think, a bit differently
than what you've seen before.

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00:10:23,900 --> 00:10:26,700
Again, very much
from the standpoint

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of experimental methods
and hypothesis testing.

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So we'll cover
protein synthesis,

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doing a careful case
study of the ribosome.

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We'll continue with
protein folding.

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So asking the question,
after the ribosome

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synthesizes a polypeptide
chain, how does that polypeptide

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assemble into its native form?

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What happens when
proteins are misfolded?

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And then we'll move into
protein degradation,

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and we'll look at proteases
and machines that are involved

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in proteolytic degradation.

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00:11:01,640 --> 00:11:04,160
And where we'll
close the first half

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is with module four,
which is on synthases,

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or often called
assembly-line enzymology.

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And this is a different type of
template-driven polymerization

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that's involved in the
synthesis of natural products.

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And then after spring
break, Professor Stubbe

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will take over,
and the focus will

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be on cellular processes
that involve homeostasis,

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00:11:27,080 --> 00:11:29,330
metabolism, and signaling.

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00:11:29,330 --> 00:11:34,910
And so these topics will involve
cholesterol biosynthesis,

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and a type of molecule
called terpene.

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And so a third way to
make a carbon-carbon bond

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will be introduced
in this section.

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So you've heard about Claisen
and Aldol condensations

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in prior biochemistry courses,
this will be another route.

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And then, we both love
metals and biology,

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so there's a whole field
of bioinorganic chemistry,

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and it will be introduced to
you here with iron homeostasis

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00:12:01,340 --> 00:12:03,980
as a case study.

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And moving from here, and
something quite related,

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involves reactive
oxygen species.

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So I'm sure you've all heard
about these somewhere, maybe

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in the news, maybe
from your lab work.

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00:12:16,340 --> 00:12:19,340
What are these reactive
oxygen species?

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00:12:19,340 --> 00:12:20,720
Are they all reactive?

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00:12:20,720 --> 00:12:23,300
What kind of chemistry
do they do in a cell?

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How do we study that here?

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00:12:26,630 --> 00:12:30,340
And then, of course, we'll
close with a section,

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00:12:30,340 --> 00:12:36,020
a module on nucleotide and
deoxynucleotide metabolism--

250
00:12:36,020 --> 00:12:38,480
excuse me-- as
well as regulation.

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And then an integration
of course concepts.

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So we have a lot of exciting
topics and exciting things

253
00:12:44,840 --> 00:12:47,720
to tell you about.

254
00:12:47,720 --> 00:12:49,760
In terms of level
of understanding

255
00:12:49,760 --> 00:12:54,910
for this course, as I said, many
of these systems are complex.

256
00:12:54,910 --> 00:12:58,180
We're going to look at huge
macromolecular machines,

257
00:12:58,180 --> 00:13:00,640
and multi-step processes.

258
00:13:00,640 --> 00:13:03,160
This is a biochemistry
course, and we

259
00:13:03,160 --> 00:13:06,640
are interested in molecular
level, in addition

260
00:13:06,640 --> 00:13:08,480
to this big picture.

261
00:13:08,480 --> 00:13:13,440
And so things to keep in mind
when thinking about structure.

262
00:13:13,440 --> 00:13:15,640
You need to think
about the amino acids,

263
00:13:15,640 --> 00:13:20,290
and please review these
if you're a bit rusty.

264
00:13:20,290 --> 00:13:24,640
So to know the side chains,
PKAs, et cetera, that's

265
00:13:24,640 --> 00:13:27,670
all important to have in mind.

266
00:13:27,670 --> 00:13:29,710
What are the protein folds?

267
00:13:29,710 --> 00:13:31,960
What are the arrangements
of these macromolecular

268
00:13:31,960 --> 00:13:35,680
assemblies, and how
do we study that?

269
00:13:35,680 --> 00:13:37,990
In terms of
reactivity, we'll see

270
00:13:37,990 --> 00:13:40,520
bond-breaking and
bond-forming reactions.

271
00:13:40,520 --> 00:13:44,140
So again, we need to think about
things like PKAs, nucleophiles,

272
00:13:44,140 --> 00:13:46,060
and electrophiles.

273
00:13:46,060 --> 00:13:49,090
If you need to brush up,
organic chemistry textbook

274
00:13:49,090 --> 00:13:52,800
or biochemistry textbook
is a good place to go.

275
00:13:52,800 --> 00:13:56,410
And then something to
keep in mind is dynamics.

276
00:13:56,410 --> 00:14:00,220
So the macromolecular structures
and enzymes and proteins

277
00:14:00,220 --> 00:14:01,900
we'll look at are dynamic.

278
00:14:01,900 --> 00:14:05,140
Often we only have a static
picture or some number

279
00:14:05,140 --> 00:14:06,910
of static pictures.

280
00:14:06,910 --> 00:14:09,310
But there's
conformational change,

281
00:14:09,310 --> 00:14:11,320
transient binding
occurs, and we always

282
00:14:11,320 --> 00:14:13,220
need to think about kinetics.

283
00:14:13,220 --> 00:14:15,430
So these are things just
to keep in mind when you're

284
00:14:15,430 --> 00:14:18,700
reading and questioning to
yourself about any given

285
00:14:18,700 --> 00:14:21,670
system here.

286
00:14:21,670 --> 00:14:25,570
So what about
experimental methods?

287
00:14:25,570 --> 00:14:31,180
This is just another topic to
go over in this course overview.

288
00:14:31,180 --> 00:14:36,010
So there's many methods
that come up in 5.08.

289
00:14:36,010 --> 00:14:38,530
And we don't expect
that you have

290
00:14:38,530 --> 00:14:42,700
knowledge of any or all of
these at the stage of starting

291
00:14:42,700 --> 00:14:44,200
the course.

292
00:14:44,200 --> 00:14:47,350
The difficulty that comes up
is that we can't introduce

293
00:14:47,350 --> 00:14:49,780
all of these methods
to you at once

294
00:14:49,780 --> 00:14:54,940
in a level of detail that's
needed for everything we do.

295
00:14:54,940 --> 00:14:57,940
OK, so what will happen
is that if methods come up

296
00:14:57,940 --> 00:15:00,750
in problem sets that
haven't yet been addressed,

297
00:15:00,750 --> 00:15:02,920
we'll give you enough
background information

298
00:15:02,920 --> 00:15:05,320
in the problems that
material, such that you

299
00:15:05,320 --> 00:15:09,700
can think about the
questions and answer them.

300
00:15:09,700 --> 00:15:12,400
And we'll let you know
when a method comes up.

301
00:15:12,400 --> 00:15:14,530
You know, you'll hear
this in recitation x,

302
00:15:14,530 --> 00:15:17,560
or we'll talk about
it more in class.

303
00:15:17,560 --> 00:15:20,110
So right now, what
I'd like to do

304
00:15:20,110 --> 00:15:22,180
is just go over a
few of the methods

305
00:15:22,180 --> 00:15:26,110
that you're going to
see multiple times.

306
00:15:26,110 --> 00:15:31,660
And the thing to keep in mind
is that the context in which

307
00:15:31,660 --> 00:15:34,690
these methods are
being used may differ,

308
00:15:34,690 --> 00:15:39,270
but the underlying
principles are the same.

309
00:15:39,270 --> 00:15:42,340
And we choose methods
that are being used today,

310
00:15:42,340 --> 00:15:43,780
and are important.

311
00:15:43,780 --> 00:15:46,300
Some of these were developed
decades ago, some of these

312
00:15:46,300 --> 00:15:49,480
are very, very new,
and hot off the press.

313
00:15:49,480 --> 00:15:53,290
So if it's an older paper,
please don't brush it off as,

314
00:15:53,290 --> 00:15:55,300
like, oh, this is old.

315
00:15:55,300 --> 00:15:57,940
And so, you know, it's not new.

316
00:15:57,940 --> 00:16:01,510
We're all really excited by
technology and everything here,

317
00:16:01,510 --> 00:16:05,020
but many of these older
methods are robust,

318
00:16:05,020 --> 00:16:07,630
and used all the time here.

319
00:16:07,630 --> 00:16:11,280
So what are some
methods and tools

320
00:16:11,280 --> 00:16:13,030
that we'll have under our belt?

321
00:16:18,880 --> 00:16:21,400
The first to point out
are methods involved

322
00:16:21,400 --> 00:16:23,050
in macromolecular structure.

323
00:16:34,900 --> 00:16:37,450
So we care a lot
about structure,

324
00:16:37,450 --> 00:16:40,090
because we need
structural understanding

325
00:16:40,090 --> 00:16:44,050
to be able to comprehend
how these systems work.

326
00:16:44,050 --> 00:16:46,690
And so one method
we'll see a lot--

327
00:16:46,690 --> 00:16:49,420
and you'll discuss in
recitation this week--

328
00:16:49,420 --> 00:16:51,400
is x-ray crystallography.

329
00:16:58,270 --> 00:17:01,420
And in addition, a method that
will come up quite a bit--

330
00:17:01,420 --> 00:17:03,790
and we'll see both of these
in the initial discussions

331
00:17:03,790 --> 00:17:05,500
of the ribosome--

332
00:17:05,500 --> 00:17:07,050
is electron microscopy.

333
00:17:15,640 --> 00:17:18,990
And another method to be aware
of-- and if you're curious,

334
00:17:18,990 --> 00:17:22,000
talk to your TA, Shiva--

335
00:17:22,000 --> 00:17:23,579
is NMR.

336
00:17:23,579 --> 00:17:27,940
OK, so NMR has a
lot of applications

337
00:17:27,940 --> 00:17:30,820
here within
biological chemistry,

338
00:17:30,820 --> 00:17:33,700
but we won't discuss that.

339
00:17:33,700 --> 00:17:37,930
What can go along with
methods is bioinformatics.

340
00:17:48,100 --> 00:17:51,970
So how many of you
have used BLAST?

341
00:17:51,970 --> 00:17:53,770
How many of you know
what BLAST stands for?

342
00:17:56,277 --> 00:17:58,110
AUDIENCE: Basic Local
Alignment Search Tool.

343
00:17:58,110 --> 00:18:01,060
ELIZABETH NOLAN: Yeah, Basic
Local Alignment Search Tool.

344
00:18:01,060 --> 00:18:03,960
So what does this let you do?

345
00:18:07,880 --> 00:18:11,510
It lets you find regions of
similarity between sequences,

346
00:18:11,510 --> 00:18:13,970
whether that's
amino acid sequence,

347
00:18:13,970 --> 00:18:16,140
a nucleotide sequence.

348
00:18:16,140 --> 00:18:20,030
And you can use that information
to make hypotheses and design

349
00:18:20,030 --> 00:18:23,090
experiments there.

350
00:18:23,090 --> 00:18:24,020
So that will come up.

351
00:18:26,720 --> 00:18:30,710
I have additional methods
and possibilities.

352
00:18:30,710 --> 00:18:32,090
What about fluorescence?

353
00:18:41,350 --> 00:18:44,890
So how many of you have
done an experiment that

354
00:18:44,890 --> 00:18:49,360
involves fluorescence, either
in lab, or in your research?

355
00:18:49,360 --> 00:18:53,890
How many, did that involve
a fluorescent protein?

356
00:18:53,890 --> 00:18:57,300
What about a small molecule?

357
00:18:57,300 --> 00:18:59,530
Yeah.

358
00:18:59,530 --> 00:19:00,400
That's fluorescent.

359
00:19:00,400 --> 00:19:03,130
So have you thought
about why the protein was

360
00:19:03,130 --> 00:19:07,540
used, versus maybe why a
small molecule, and what

361
00:19:07,540 --> 00:19:09,490
are inherent strengths
and limitations or one

362
00:19:09,490 --> 00:19:13,180
or the other, depending
what you want to do?

363
00:19:13,180 --> 00:19:17,530
So fluorescence is used in
many, many different contexts.

364
00:19:17,530 --> 00:19:21,410
We can think about proteins
like green fluorescent protein,

365
00:19:21,410 --> 00:19:23,400
we can think about
using small molecules.

366
00:19:28,750 --> 00:19:32,170
And we like fluorescence
because it allows us to see.

367
00:19:32,170 --> 00:19:36,490
We can get visual information.

368
00:19:36,490 --> 00:19:39,890
And so, where fluorescence will
first come up in this class

369
00:19:39,890 --> 00:19:42,280
is with the ribosome.

370
00:19:42,280 --> 00:19:44,830
And in recitation
week two, there'll

371
00:19:44,830 --> 00:19:48,370
be some discussion about using
small molecule fluorophores

372
00:19:48,370 --> 00:19:51,310
to label tRNAs, and
using fluorescence

373
00:19:51,310 --> 00:19:54,980
as a readout of steps in
the translation process.

374
00:19:54,980 --> 00:19:58,160
And there's a lot of
considerations and caveats

375
00:19:58,160 --> 00:19:58,660
to that.

376
00:19:58,660 --> 00:19:59,890
Do we have a pizza delivery?

377
00:19:59,890 --> 00:20:01,840
Thank goodness no.

378
00:20:01,840 --> 00:20:04,220
Often in this class, we get
pizza deliveries for someone

379
00:20:04,220 --> 00:20:04,720
else.

380
00:20:04,720 --> 00:20:07,520
I didn't know if that's
already starting.

381
00:20:07,520 --> 00:20:09,760
Yeah, yeah.

382
00:20:09,760 --> 00:20:13,900
We'll also see GFP being used
in the proteasome section

383
00:20:13,900 --> 00:20:15,080
for degradation.

384
00:20:15,080 --> 00:20:17,640
So a folded protein
has fluorescence,

385
00:20:17,640 --> 00:20:21,227
a degraded protein does not.

386
00:20:21,227 --> 00:20:22,060
What about kinetics?

387
00:20:30,050 --> 00:20:35,230
So what different types of
kinetic studies can be done?

388
00:20:35,230 --> 00:20:38,420
So what do we all hear about
in introductory biochemistry

389
00:20:38,420 --> 00:20:38,920
class?

390
00:20:46,630 --> 00:20:47,570
Pardon?

391
00:20:47,570 --> 00:20:48,550
AUDIENCE: [INAUDIBLE].

392
00:20:48,550 --> 00:20:50,717
ELIZABETH NOLAN: Yeah,
steady state kinetics, right?

393
00:20:50,717 --> 00:20:51,830
Turnover.

394
00:20:51,830 --> 00:20:58,510
So we have steady state, which
I encourage you to review

395
00:20:58,510 --> 00:21:02,080
Michaelis-Mentin Kinetics here.

396
00:21:02,080 --> 00:21:05,500
And you'll also be
introduced in the first weeks

397
00:21:05,500 --> 00:21:08,500
of this course, and
especially recitation three--

398
00:21:08,500 --> 00:21:11,710
so recitation two is going
to build up to this--

399
00:21:11,710 --> 00:21:12,910
pre-steady state kinetics.

400
00:21:24,870 --> 00:21:29,550
So here, you've heard
about this in 5.07

401
00:21:29,550 --> 00:21:33,030
or another course,
introductory course.

402
00:21:33,030 --> 00:21:37,650
And we're looking at multiple
turnover of an enzyme.

403
00:21:41,260 --> 00:21:45,260
And these experiments are set
up with an excess of substrate,

404
00:21:45,260 --> 00:21:47,170
right, in order to
afford conditions

405
00:21:47,170 --> 00:21:48,430
that allow multiple turnover.

406
00:21:57,440 --> 00:22:00,620
So there's formation of an
enzyme substrate complex,

407
00:22:00,620 --> 00:22:02,750
and then there's
product formation.

408
00:22:02,750 --> 00:22:04,610
So review as needed.

409
00:22:04,610 --> 00:22:09,230
So what about pre-steady
state kinetics?

410
00:22:09,230 --> 00:22:13,990
How many of you are
familiar with this method?

411
00:22:13,990 --> 00:22:14,740
Not so much.

412
00:22:14,740 --> 00:22:16,225
So what does the name suggest?

413
00:22:20,084 --> 00:22:20,584
Pardon?

414
00:22:25,682 --> 00:22:27,890
JOANNE STUBBE: So I'm deaf,
you have to speak louder.

415
00:22:27,890 --> 00:22:29,680
ELIZABETH NOLAN:
Yeah, we're both deaf.

416
00:22:29,798 --> 00:22:31,090
JOANNE STUBBE: I'm really deaf.

417
00:22:31,090 --> 00:22:33,310
So if you want to say
something, so I can hear it.

418
00:22:35,880 --> 00:22:36,742
Speak up.

419
00:22:36,742 --> 00:22:38,700
AUDIENCE: Yeah, maybe
observing single molecule

420
00:22:38,700 --> 00:22:40,180
by some spectroscopy.

421
00:22:40,180 --> 00:22:42,310
ELIZABETH NOLAN: Yeah, a
single turnover, maybe, I

422
00:22:42,310 --> 00:22:43,560
think is what.

423
00:22:43,560 --> 00:22:45,460
If we're having
multiple turnovers here

424
00:22:45,460 --> 00:22:47,380
in the steady state, right?

425
00:22:47,380 --> 00:22:49,150
If we're before
the steady state,

426
00:22:49,150 --> 00:22:51,700
what does that mean, right?

427
00:22:51,700 --> 00:22:53,530
It means we're in
the initial, really

428
00:22:53,530 --> 00:22:56,950
initial part of this
reaction, where we're looking

429
00:22:56,950 --> 00:22:58,435
at a single turnover here.

430
00:23:02,130 --> 00:23:03,255
And how would you do this?

431
00:23:06,040 --> 00:23:11,610
Basically, you look with subs
having limiting substrate

432
00:23:11,610 --> 00:23:14,880
rather than excess substrate.

433
00:23:14,880 --> 00:23:20,040
And this is just to
give a little prelude

434
00:23:20,040 --> 00:23:23,850
in terms of thinking
about experimental design.

435
00:23:23,850 --> 00:23:27,735
So here, look at the first
moments of a reaction.

436
00:23:34,920 --> 00:23:39,168
So what type of
time scale is that?

437
00:23:39,168 --> 00:23:40,032
AUDIENCE: Small.

438
00:23:40,032 --> 00:23:41,710
ELIZABETH NOLAN: Yeah, small.

439
00:23:41,710 --> 00:23:46,390
Maybe a millisecond time
scale, compared to a timescale

440
00:23:46,390 --> 00:23:48,850
of seconds or minutes.

441
00:23:48,850 --> 00:23:50,120
So what does that mean?

442
00:23:50,120 --> 00:23:53,400
It means you need some
different experimental setup.

443
00:23:53,400 --> 00:23:55,420
You can't do pre-steady
state kinetics

444
00:23:55,420 --> 00:23:58,480
in the way we've done steady
state kinetics, say in a lab

445
00:23:58,480 --> 00:24:00,420
class for instance.

446
00:24:00,420 --> 00:24:02,570
So you need a special apparatus.

447
00:24:02,570 --> 00:24:06,700
And what does it let you see?

448
00:24:06,700 --> 00:24:11,460
Here you're looking at multiple
turnover, products forming.

449
00:24:11,460 --> 00:24:15,430
You know, here in the early
stages, what can you see?

450
00:24:15,430 --> 00:24:18,970
Maybe intermediate formation.

451
00:24:18,970 --> 00:24:22,690
And why might that be important
for thinking about mechanism?

452
00:24:22,690 --> 00:24:25,360
So those will come up in the
first weeks of recitation.

453
00:24:28,380 --> 00:24:33,240
Another topic that will
come up, and is something

454
00:24:33,240 --> 00:24:37,470
that you always need to
think about, and relates

455
00:24:37,470 --> 00:24:42,360
to integrity of materials,
is that of purification.

456
00:24:42,360 --> 00:24:45,240
So how are proteins purified.

457
00:24:45,240 --> 00:24:48,630
For studying the
ribosome, how do we

458
00:24:48,630 --> 00:24:52,770
get ribosomes that are
pure and are correct?

459
00:24:52,770 --> 00:24:55,680
Or what if you'd like to
use a mutant ribosome?

460
00:24:55,680 --> 00:24:59,610
How does that get generated?

461
00:24:59,610 --> 00:25:09,390
So here, you can talk
about ribosome or protein

462
00:25:09,390 --> 00:25:12,530
purification.

463
00:25:12,530 --> 00:25:17,610
And so, I'll present to you on
ribosomes and mutant ribosomes

464
00:25:17,610 --> 00:25:19,780
in week four of recitation.

465
00:25:19,780 --> 00:25:23,340
And this topic more
generally of proteins

466
00:25:23,340 --> 00:25:26,610
will come up in passing
again and again.

467
00:25:26,610 --> 00:25:30,860
So how many of you have
purified a protein?

468
00:25:30,860 --> 00:25:31,360
Many.

469
00:25:31,360 --> 00:25:33,370
How many of you used
an affinity tag?

470
00:25:36,800 --> 00:25:38,950
So are they the answer
to all problems?

471
00:25:41,920 --> 00:25:42,420
No.

472
00:25:42,420 --> 00:25:46,860
They can be a huge help, but
they can also be problematic

473
00:25:46,860 --> 00:25:48,480
in one way or another, right?

474
00:25:48,480 --> 00:25:50,790
So with the ribosome
we'll look at a case

475
00:25:50,790 --> 00:25:53,310
where there was really
some elegant work done

476
00:25:53,310 --> 00:25:57,960
using an affinity tag
approach to allow researchers

477
00:25:57,960 --> 00:26:00,510
to obtain new ribosomes.

478
00:26:00,510 --> 00:26:02,760
We'll also, though, talk
about the limitations

479
00:26:02,760 --> 00:26:04,732
of that type of
methodology, and the things

480
00:26:04,732 --> 00:26:06,690
you need to think about
if you're doing protein

481
00:26:06,690 --> 00:26:11,100
biochemistry, and how a tag
may affect your experiments

482
00:26:11,100 --> 00:26:14,370
and data there.

483
00:26:14,370 --> 00:26:29,470
In addition, to think
about is assay development,

484
00:26:29,470 --> 00:26:30,580
and analytical methods.

485
00:26:40,460 --> 00:26:44,630
And so there will be many
different types of assays that

486
00:26:44,630 --> 00:26:47,360
are presented in this course.

487
00:26:47,360 --> 00:26:49,610
And something just
to think about--

488
00:26:49,610 --> 00:26:55,180
how do you develop the
right assay, and what

489
00:26:55,180 --> 00:26:59,170
are all the considerations?

490
00:26:59,170 --> 00:27:03,310
How do you know your assay is
a good one for the question you

491
00:27:03,310 --> 00:27:05,500
want to address there?

492
00:27:05,500 --> 00:27:08,510
This is actually
really complicated.

493
00:27:08,510 --> 00:27:11,860
And so there'll be
some case studies

494
00:27:11,860 --> 00:27:14,050
that come up in the course,
but just more broadly

495
00:27:14,050 --> 00:27:14,710
to think about.

496
00:27:14,710 --> 00:27:18,250
So often in lab classes,
you may have an assay,

497
00:27:18,250 --> 00:27:21,550
but you might not be aware of
all of the considerations that

498
00:27:21,550 --> 00:27:27,430
went into actually developing
that assay such that it works.

499
00:27:27,430 --> 00:27:29,410
And then there's the
analytical methods

500
00:27:29,410 --> 00:27:32,020
that are used,
either for analyzing

501
00:27:32,020 --> 00:27:34,790
assay data or other data.

502
00:27:34,790 --> 00:27:37,540
And again, these have
strengths and limitations.

503
00:27:37,540 --> 00:27:47,500
Just some that will come
up, to present western blots

504
00:27:47,500 --> 00:27:49,070
and immunoprecipitation.

505
00:27:57,120 --> 00:27:59,670
So these methods
involve antibodies,

506
00:27:59,670 --> 00:28:05,490
and so we need to think about
the antibodies themselves here.

507
00:28:05,490 --> 00:28:06,660
Radioactivity.

508
00:28:12,670 --> 00:28:15,450
OK, how does this work?

509
00:28:15,450 --> 00:28:19,040
Why do biochemists like to
use radioactivity and assay

510
00:28:19,040 --> 00:28:19,850
development?

511
00:28:19,850 --> 00:28:23,450
And how to think about this
productively and correctly.

512
00:28:23,450 --> 00:28:31,160
So should you be afraid
of iron-55, yes or no?

513
00:28:31,160 --> 00:28:34,160
How does that exposure compare
to being in an airplane,

514
00:28:34,160 --> 00:28:36,500
for instance.

515
00:28:36,500 --> 00:28:39,500
Seriously, because there's
a lot of fear associated

516
00:28:39,500 --> 00:28:42,500
with radioactivity that may
or may not be well-founded,

517
00:28:42,500 --> 00:28:44,930
depending on what you're doing.

518
00:28:44,930 --> 00:28:47,840
And so this gives us
a lot of sensitivity.

519
00:28:47,840 --> 00:28:50,750
And JoAnne will talk in
week two of recitation

520
00:28:50,750 --> 00:28:54,170
about radioactivity, and
designing experiments

521
00:28:54,170 --> 00:28:58,160
that use this as a read out.

522
00:28:58,160 --> 00:28:59,150
What else?

523
00:28:59,150 --> 00:29:00,740
So affinity measurements.

524
00:29:10,380 --> 00:29:14,580
OK, so dissociation constants,
or affinity constants,

525
00:29:14,580 --> 00:29:17,460
how are these measured?

526
00:29:17,460 --> 00:29:20,790
When reading the literature,
is the value a good one,

527
00:29:20,790 --> 00:29:25,410
or a not-so-good one, and how
can you make that distinction?

528
00:29:25,410 --> 00:29:26,870
Mass spec and proteomics.

529
00:29:35,740 --> 00:29:37,950
So these will be in the
later half of the class--

530
00:29:37,950 --> 00:29:41,020
I believe recitations
11 and 12--

531
00:29:41,020 --> 00:29:42,700
and many others.

532
00:29:42,700 --> 00:29:44,800
And we're introducing
CRISPR this year,

533
00:29:44,800 --> 00:29:48,880
in the context of the
cholesterol unit as well.

534
00:29:48,880 --> 00:29:52,120
So as I said, we can't take
care of all of these methods

535
00:29:52,120 --> 00:29:53,670
immediately.

536
00:29:53,670 --> 00:29:55,420
We'll let you know
when they're coming up,

537
00:29:55,420 --> 00:30:00,370
when you need to know more
details about them as we

538
00:30:00,370 --> 00:30:05,320
go through the
course here for that.

539
00:30:05,320 --> 00:30:08,560
So we can get started.

540
00:30:08,560 --> 00:30:11,410
And in the last
few minutes, what

541
00:30:11,410 --> 00:30:16,900
I'll do is just give you a brief
overview of the macromolecular

542
00:30:16,900 --> 00:30:22,390
machines we'll look at through
modules one through three.

543
00:30:25,190 --> 00:30:28,020
And basically, what
is the big picture?

544
00:30:28,020 --> 00:30:30,890
And then we're going
to break that down

545
00:30:30,890 --> 00:30:34,110
into looking at
individual components.

546
00:30:34,110 --> 00:30:44,210
So if we think about the
lifecycle of a protein,

547
00:30:44,210 --> 00:30:49,520
basically, we'll fast forward to
having mRNA from transcription

548
00:30:49,520 --> 00:30:52,250
of the genetic code.

549
00:30:52,250 --> 00:30:55,040
And then we have the
macromolecular machine,

550
00:30:55,040 --> 00:31:02,950
the ribosome that allows for
translation of this method

551
00:31:02,950 --> 00:31:04,925
message to give us
a polypeptide chain.

552
00:31:10,070 --> 00:31:14,180
So some linear sequence
of amino acids.

553
00:31:14,180 --> 00:31:16,550
And then what happens?

554
00:31:16,550 --> 00:31:18,770
We need to get from
a polypeptide chain

555
00:31:18,770 --> 00:31:20,750
to some functional unit.

556
00:31:20,750 --> 00:31:26,390
And so there's a whole
number of interesting players

557
00:31:26,390 --> 00:31:29,600
that are involved
in protein folding.

558
00:31:29,600 --> 00:31:40,470
So we have folding, which
is enabled by chaperones,

559
00:31:40,470 --> 00:31:44,560
is what we call these proteins
that facilitate folding.

560
00:31:44,560 --> 00:31:50,230
And that's going to give
us some structure that

561
00:31:50,230 --> 00:31:56,930
has some function here.

562
00:31:56,930 --> 00:32:00,920
And this protein has some
lifetime in the cell.

563
00:32:00,920 --> 00:32:03,200
So at some time,
for some reason,

564
00:32:03,200 --> 00:32:07,220
it will be time for this
protein to get degraded.

565
00:32:07,220 --> 00:32:09,620
In which case, we
need machinery that

566
00:32:09,620 --> 00:32:14,330
will facilitate the process to
break down this folded protein

567
00:32:14,330 --> 00:32:16,190
into smaller
fragments-- whether that

568
00:32:16,190 --> 00:32:19,700
be individual amino acids,
or short polypeptide chains

569
00:32:19,700 --> 00:32:22,520
of seven to eight amino acids.

570
00:32:22,520 --> 00:32:33,990
So from here, we
have degradation

571
00:32:33,990 --> 00:32:38,070
to give us small fragments.

572
00:32:38,070 --> 00:32:44,970
And the players here are
proteases and chambers of doom,

573
00:32:44,970 --> 00:32:46,620
one of which is the proteasome.

574
00:32:51,290 --> 00:32:52,850
And actually, I
forgot to mention

575
00:32:52,850 --> 00:32:56,690
there will be a second
guest lecturer in recitation

576
00:32:56,690 --> 00:32:59,570
this year, Reuben
Saunders, who is a senior,

577
00:32:59,570 --> 00:33:01,670
and does research
in the Sauer Lab

578
00:33:01,670 --> 00:33:05,150
on one of these chambers
of doom, called ClpXP.

579
00:33:05,150 --> 00:33:08,000
And so he'll present on
single molecule methods,

580
00:33:08,000 --> 00:33:11,570
and fluorescence methods to
study how this degradation

581
00:33:11,570 --> 00:33:12,310
chamber works.

582
00:33:12,310 --> 00:33:13,790
So that will be really exciting.

583
00:33:13,790 --> 00:33:16,950
He was a student in our
course two years ago.

584
00:33:16,950 --> 00:33:18,750
So let's just take a look.

585
00:33:18,750 --> 00:33:20,480
We have the ribosome here.

586
00:33:23,290 --> 00:33:27,310
What are the structural features
of this macromolecular machine,

587
00:33:27,310 --> 00:33:29,140
and how does it do its job?

588
00:33:29,140 --> 00:33:33,100
We'll look at a number of
seminal studies that were done.

589
00:33:33,100 --> 00:33:37,300
And it is truly
fascinating and incredible.

590
00:33:37,300 --> 00:33:39,670
What about protein folding?

591
00:33:39,670 --> 00:33:43,730
So look at this
macromolecular machine here,

592
00:33:43,730 --> 00:33:47,650
GroEL, GroES, look
at how big this is.

593
00:33:47,650 --> 00:33:56,320
So how does this chaperone
allow some nascent polypeptide

594
00:33:56,320 --> 00:33:58,750
that's unfolded or
partially folded

595
00:33:58,750 --> 00:34:01,510
to obtain its native structure?

596
00:34:01,510 --> 00:34:04,210
And there is many details
in this depiction here

597
00:34:04,210 --> 00:34:06,160
that probably
aren't apparent yet.

598
00:34:06,160 --> 00:34:12,610
But by the time we're done with
module two, it will be there.

599
00:34:12,610 --> 00:34:14,929
Protein degradation.

600
00:34:14,929 --> 00:34:22,120
So here is just a cartoon-type
depiction of a chamber of doom

601
00:34:22,120 --> 00:34:26,920
and its accessory protein
from E. coli, ClpZ, ClpP.

602
00:34:26,920 --> 00:34:29,290
So look, we have a
folded protein here, it's

603
00:34:29,290 --> 00:34:33,340
a beta barrel, our friend
GFP that emits green light.

604
00:34:33,340 --> 00:34:37,719
And somehow, this protein
gets threaded through ClpX,

605
00:34:37,719 --> 00:34:39,400
enters this chamber--

606
00:34:39,400 --> 00:34:41,560
which has multiple
protease active sites--

607
00:34:41,560 --> 00:34:44,440
and that protein
gets all degraded.

608
00:34:44,440 --> 00:34:46,159
So how does this work?

609
00:34:46,159 --> 00:34:50,620
How did ClpX and P work
together to allow degradation

610
00:34:50,620 --> 00:34:53,920
of this condemned protein?

611
00:34:53,920 --> 00:34:56,350
And then finally, where
I'll close is on something

612
00:34:56,350 --> 00:34:59,140
I think a little bit
different for most everyone,

613
00:34:59,140 --> 00:35:02,590
and it's a type of
template-driven polymerization

614
00:35:02,590 --> 00:35:05,170
involved in the synthesis
of small molecules

615
00:35:05,170 --> 00:35:07,420
like penicillins
and erythromycins.

616
00:35:07,420 --> 00:35:09,880
So these are antibiotics.

617
00:35:09,880 --> 00:35:13,420
So how do we get at
molecules like these

618
00:35:13,420 --> 00:35:16,390
from simple amino
acid precursors,

619
00:35:16,390 --> 00:35:20,860
or precursors like those you've
seen in fatty acid biosynthesis

620
00:35:20,860 --> 00:35:21,640
here?

621
00:35:21,640 --> 00:35:25,343
And often, these are
described as assembly lines.

622
00:35:25,343 --> 00:35:27,760
And something we'll just need
to keep in mind in this unit

623
00:35:27,760 --> 00:35:31,090
is, are these proteins really
acting like an assembly line,

624
00:35:31,090 --> 00:35:34,690
or is this just a way to help
us think about the templates

625
00:35:34,690 --> 00:35:36,160
and what's going on here?

626
00:35:36,160 --> 00:35:38,290
So that's where we'll close.

627
00:35:38,290 --> 00:35:41,350
OK, so with that I'll
finish up, and on Friday

628
00:35:41,350 --> 00:35:43,780
we'll begin with
looking at the structure

629
00:35:43,780 --> 00:35:46,740
of the prokaryotic ribosome.